Methods and Devices for Using Mucolytic Agents Including N-Acetyl Cysteine (NAC)

ABSTRACT

Devices and methods incorporate mucolytic agents into a point-of-care testing device. The sample is loaded, and then the sample travels until it encounters one or more lysis agents and/or mucolytic agents. The mucolytic agent is preferably pre-loaded onto the collection device. In a preferred embodiment, the mucolytic agent is localized between the sample application zone and the conjugate zone. In embodiments with a sample compressor, one or more mucolytic agents may be pre-loaded and dried on the sample compressor, the sample collector, in various locations on the test strip, or in the running buffer.

REFERENCE TO RELATED APPLICATIONS

This application claims one or more inventions which were disclosed inProvisional Application No. 61/481,907, filed May 3, 2011, entitled“METHODS AND DEVICES FOR USING MUCOLYTIC AGENTS INCLUDING N-ACETYLCYSTEINE (NAC)”. The benefit under 35 USC §119(e) of the United Statesprovisional application is hereby claimed, and the aforementionedapplication is hereby incorporated herein by reference.

This application is also a continuation in-part of co-pendingapplication Ser. No. 12/502,662, filed Jul. 14, 2009, entitled “IN SITULYSIS OF CELLS IN LATERAL FLOW IMMUNOASSAYS”, which claims one or moreinventions which were disclosed in Provisional Application No.61/080,879, filed Jul. 15, 2008, entitled “LATERAL FLOW NUCLEIC ACIDDETECTOR”, Provisional Application No. 61/098,935, filed Sep. 22, 2008,entitled “IN SITU LYSIS OF CELLS IN LATERAL FLOW IMMUNOASSAYS”, andProvisional Application No. 61/179,059, filed May 18, 2009, entitled“METHOD AND DEVICE FOR COMBINED DETECTION OF VIRAL AND BACTERIALINFECTIONS”, and which is a continuation-in-part application ofapplication Ser. No. 12/469,207, filed May 20, 2009, entitled“NANOPARTICLES IN DIAGNOSTIC TESTS”, which claimed priority fromProvisional Application No. 61/071,833, filed May 20, 2008, entitled“NANOPARTICLES IN DIAGNOSTIC TESTS” and application Ser. No. 12/481,631,filed Jun. 10, 2009, entitled “COMBINED VISUAL/FLUORESCENCE ANALYTEDETECTION TEST”, which claimed priority from Provisional Application No.61/060,258, filed Jun. 10, 2008, entitled “COMBINED VISUAL/FLUORESCENCEANALYTE DETECTION TEST”.

This application is also a continuation in-part of co-pendingapplication Ser. No. 12/958,454, filed Dec. 2, 2010, entitled“MULTIPLANAR LATERAL FLOW ASSAY WITH SAMPLE COMPRESSOR”, which claimsone or more inventions which were disclosed in Provisional ApplicationNo. 61/266,641, filed Dec. 4, 2009, entitled “LATERAL FLOW NUCLEIC ACIDDETECTOR”, Provisional Application No. 61/331,966, filed May 6, 2010,entitled “MULTIPLANAR LATERAL FLOW ASSAY WITH SAMPLE COMPRESSOR”,Provisional Application No. 61/352,093, filed Jun. 7, 2010, entitled“LATERAL FLOW ASSAYS”, and Provisional Application No. 61/392,981, filedOct. 14, 2010, entitled “MULTIPLANAR LATERAL FLOW ASSAY WITH SAMPLECOMPRESSOR”.

The benefit under 35 USC §119(e) of the United States provisionalapplications is hereby claimed, and the aforementioned applications arehereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention pertains to the field of lateral flow assays. Moreparticularly, the invention pertains to in situ lysis and mucolysis ofsamples in lateral flow assays.

2. Description of Related Art

Lateral flow immunoassays combine the reagents and the process steps ofmore general immunoassays into an improved assay. This enablessingle-step, point-of care testing (POCT) and provides a sensitive andrapid means for detection of target molecules. Lateral flow immunoassaysare available for a wide array of target analytes and can be designedfor sandwich or competitive test formats. Generally high molecularweight analytes with several epitopes are analyzed in a sandwich formatwhereas small molecules representing only one epitope are detected bymeans of a competitive assay. The first lateral flow assays tested forhuman chorionic gonadotropin (hCG). Today commercially available testsmonitor ovulation, detect infectious disease organisms, analyze drugs ofabuse and measure other analytes important to human physiology. Productshave also been introduced for veterinary testing, environmental testing,and product monitoring.

U.S. Pat. No. 5,714,341 discloses a lateral flow immunoassay for HIVspecific antibodies in saliva samples. A saliva sample is diluted in asample buffer, and a lateral flow immunoassay is dipped into the dilutedsaliva sample, again enabling point-of-care testing with rapid results.

German Patent DE19622503 discloses a lateral flow immunoassay forillegal narcotics in saliva and sweat.

There is a need for still simpler-to-use and more rapid lateral flowimmunoassays suitable for time-sensitive and cost-sensitive clinicalsettings. This need is most acute in situations where the sample typeand target analyte necessitate a sample preparation step. This may occurwhen an analyte is not readily presented within a sample and a separatelysis step is necessary to free the analyte for efficient presentation.Such an assay may need to directly test for analytes in human bodyfluids, including analytes which may be protected within complexes orbehind membranes including cellular or mucosal membranes.

As an example, fever is a common cause of childhood visits to urgentcare centers for both family practice and pediatric offices. Mostcommonly, this relates to either a respiratory infection orgastroenteritis. The high incidence of fever in children and theprecautious administration of unnecessary antibiotics is reason todevelop a rapid screening test for biomarkers that distinguish viralfrom bacterial infections.

The efficiency and even the probability of success of a givenimmunoassay will depend on the initial presentation of any antigens tobe detected. Antigens and other targets must be accessible to antibodiesof an assay. Access can be impacted if most or all of the availableantigen is masked in a complex or is inaccessible behind a cellmembrane, e.g. in a cell's cytoplasm. In these situations, a viable andefficient assay may need to include a lysis step designed to make anantigen accessible, either by breaking up a complex to unmask componentsor by removing barriers such as a cell wall, a membrane of a cell ororganelle, or a coat of a virus. However, such an added lysis step maycomplicate and delay an assay, even causing it to be too complex or timeconsuming for practical operation in a clinical setting.

In order to detect analytes protected within a complex of molecules orbehind a membrane or other barrier, one approach in the immunoassayfield is to lyse the complex or barrier and extract the analyte ofinterest prior to performing the immunoassay. When the barrier is a cellwall or cell membrane, the cells can be erythrocytes, leukocytes,epidermal, viral, fungal or bacterial, and they can be normal ormalignant. Traditionally, a required lysis step is accomplished prior toand physically separate from the desired immunoassay, as a samplepreparation step.

Practical operation in point-of care testing means that an assay needsto operate in such a manner as to report a result meeting point-of-caretesting requirements including but not limited to timeliness, accuracy,sensitivity, specificity, and ease of use. Therefore, there is a need inthe art for methods and devices that can circumvent the need for aseparate lysis or mucolysis step prior to running a lateral flow assay.

SUMMARY OF THE INVENTION

Instead of breaking down cells prior to transferring a sample to apoint-of-care testing device, the present invention includes devices andmethods that incorporate lysis and/or mucolytic agents into apoint-of-care testing device so that lysis and/or mucolysis does notneed to be conducted as a separate step. The lysis or mucolysis step isperformed on the test strip itself, as an integral part of the sampleanalysis device.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sample analysis device that may be used in embodiments ofthe present invention.

FIG. 2 shows a housing containing the strip of FIG. 1.

FIG. 3 shows a collection device for collecting a sample.

FIG. 4 shows a test kit including the sample analysis device of FIGS. 1and 2 and the collection device of FIG. 3.

FIG. 5A shows a sample analysis device including a lysis zone locatedbetween a sample application zone and a conjugate zone in an embodimentof the present invention.

FIG. 5B shows a sample analysis device including a lysis zoneoverlapping a sample application zone in an embodiment of the presentinvention.

FIG. 5C shows a sample analysis device including a lysis zoneoverlapping a conjugate zone in an embodiment of the present invention.

FIG. 5D shows a sample analysis device including a lysis zoneoverlapping a sample application zone and a conjugate zone in anembodiment of the present invention.

FIG. 6A shows a sample analysis device including a blocking zone betweena sample application zone and a conjugate zone in an embodiment of thepresent invention.

FIG. 6B shows a sample analysis device including a blocking zone betweena sample application zone and a detection zone in another embodiment ofthe present invention.

FIG. 6C shows a sample analysis device including a blocking zone betweena sample application zone and a conjugate zone in another embodiment ofthe present invention.

FIG. 6D shows a sample analysis device including a blocking zone betweena sample application zone and a detection zone in another embodiment ofthe present invention

FIG. 7A shows a sample analysis device including a mucolysis zonelocated between a sample application zone and a conjugate zone in anembodiment of the present invention.

FIG. 7B shows a sample analysis device including a mucolysis zoneoverlapping a sample application zone in an embodiment of the presentinvention.

FIG. 7C shows a sample analysis device including a mucolysis zoneoverlapping a conjugate zone in an embodiment of the present invention.

FIG. 7D shows a sample analysis device including a mucolysis zoneoverlapping a sample application zone and a conjugate zone in anembodiment of the present invention.

FIG. 8A shows a sample analysis device including a blocking zone locatedbetween a sample application zone and a conjugate zone in an embodimentof the present invention.

FIG. 8B shows a sample analysis device including a blocking zone locatedbetween a sample application zone and a detection zone in anotherembodiment of the present invention.

FIG. 8C shows a sample analysis device including a blocking zone locatedbetween a sample application zone and a conjugate zone in anotherembodiment of the present invention.

FIG. 8D shows a sample analysis device including a blocking zone locatedbetween a sample application zone and a detection zone in anotherembodiment of the present invention.

FIG. 9 shows a test strip and a sample collector in a lateral flowdevice.

FIG. 10A shows a sample compressor in an embodiment of the presentinvention.

FIG. 10B shows another sample compressor in an embodiment of the presentinvention.

FIG. 10C shows a sample collector in an embodiment of the presentinvention.

FIG. 11A shows a lateral flow test strip in an embodiment of the presentinvention.

FIG. 11B shows a full sandwich including the analyte, the conjugate, andan immobilized binding partner in an embodiment of the presentinvention.

FIG. 11C shows a lateral flow device including the test strip of FIG.11A, a sample collector, and a sample compressor in an embodiment of thepresent invention.

FIG. 12A shows a lateral flow test strip in an embodiment of the presentinvention.

FIG. 12B shows a “full” sandwich, which preferably forms before reachingthe test line, between the analyte, the labeled conjugate, and a secondtagged mobile binding partner.

FIG. 13A shows another lateral flow test strip in an embodiment of thepresent invention.

FIG. 13B shows a full sandwich including the analyte, the conjugate, anda tagged second binding partner in an embodiment of the presentinvention.

FIG. 13C shows a lateral flow device including the test strip of FIG.13A, a sample collector, and a sample compressor in an embodiment of thepresent invention.

FIG. 14A shows yet another lateral flow test strip in an embodiment ofthe present invention.

FIG. 14B shows a lateral flow device including the test strip of FIG.14A, a sample collector, and a sample compressor in another embodimentof the present invention.

FIG. 15A shows another lateral flow test strip in an embodiment of thepresent invention.

FIG. 15B shows a lateral flow device including the test strip of FIG.15A, a sample collector, and a sample compressor in another embodimentof the present invention.

FIG. 16A shows a device similar to the device of FIG. 11C except thatthe test zone is located in the sample application zone in an embodimentof the present invention.

FIG. 16B shows a device similar to the device of FIG. 13C except thatthe test zone is located in the sample application zone in an embodimentof the present invention.

FIG. 16C shows a device similar to the device of FIG. 14B except thatthe test zone is located in the sample application zone in an embodimentof the present invention.

FIG. 16D shows a device similar to the device of FIG. 15B except thatthe test zone is located in the sample application zone in an embodimentof the present invention.

FIG. 17A shows a lateral flow device in an embodiment of the presentinvention.

FIG. 17B shows another lateral flow device in an embodiment of thepresent invention.

FIG. 18 shows a vertical stack in an embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

Instead of lysing cells or treating a mucosal sample “outside” of apoint-of-care testing device, the present invention utilizes “in situlysis” or “in situ mucolysis”. The term “in situ lysis”, as used herein,describes techniques for incorporating lysis agents into a point-of-caretesting device, such as a chromatography test strip, lateral flowimmunoassay device, or other lateral flow device, so that the lysisoperation is not conducted as a separate step.

The term “in situ mucolysis” describes techniques for incorporatingmucolytic agents into a point-of-care testing device, such as achromatography test strip, lateral flow immunoassay device, or otherlateral flow device. The mucolysis step is not conducted as a separatestep.

Mucolytic agents, as defined herein, are agents that reduce theviscosity of mucus, for example by dissolving, digesting, or liquefyingthe mucus. Mucolysis, as defined herein, is the solution, digestion, orliquefaction of mucus.

Lysis agents, as defined herein, are agents that dissolve or rupturecells, for example by disrupting the cell membrane, wherebyintracellular as well as the surface proteins are exposed for betteraccess to the reagents (e.g.—antibodies in immunoassay devices).

In situ lysis and/or mucolysis offer distinct advantages over a separatelysis and/or mucolysis step. Some of these advantages are:

-   1. Higher efficiency. Cells that are lysed or mucolysed prior to    being transferred onto the device would inherently lower the    percentage of recovery. Thus, avoiding an additional transfer step    promotes efficiency and sensitivity.-   2. Higher stability. Many intra-cellular analytes are labile. By    greatly reducing the time for interaction with an antibody and    antigens in an in situ lysis or mucolysis set up, this lability can    be overcome.-   3. More rapid testing and results. In situ lysis or mucolysis    eliminates the need of separate “outside” lysing or mucolysis steps,    thus increasing the rapidity of test results in a point-of-care    testing scenario.-   4. Reducing interference. With a proper “blocking zone,” one can    block cell debris or cell bound materials from reaching an assay    reaction area. Where an analyte of interest is intra-cellular and a    protein associated with a cell wall and other cell debris needs to    be blocked, and the assay antibody is protected, then a blocking    zone downstream of the lysis and/or mucolytic agent and upstream of    an assay readout may be used to decrease interference.

“In situ lysis” and “in situ mucolysis” can also be applied to “breakingdown of the complexes,” whether they are immune complexes or boundmaterials of some kind. By lysing or mucolysing these complexes in situ,one can then measure the amount of analyte in the complexes.

In a preferred embodiment, the sample analysis device includes achromatographic test strip, e.g. a lateral flow or flow through teststrip. The test strip includes a sample application zone, a lysis zoneand/or a mucolytic zone (this zone may be incorporated into one of theother zones, overlap one of the other zones, or be a completely separatezone), a conjugate zone, and a detection zone. Preferably, the teststrip also optionally includes a waste zone, a control zone, a carrierbacking, a housing and an opening in the housing for read out of theresult. Any combinations of some or all of these elements may beincluded in the test strip. Sample analysis in the detection zone may becarried out by standard means, e.g. by an immunological, biochemical orenzymatic detection method. Preferably, the detection method includesthe use of antibodies, nucleic acids, ligands/receptors or nanoparticlescapable of specifically binding the targets, e.g. pathogens to be testedand subsequent visualization of the bound entity, e.g. by enzymaticdetection or by means of direct labeling groups, such as visible orcolored particles, dyes, magnetic particles, fluorescent orphosphorescent particles, chemiluminiscent particles, radioisotopicligands, enzymes, peptides, amino acids, colloidal particles, or beads,as is well known in the art.

Detection of the marker may be achieved in the detection zone. Thebinding molecule immobilizes the labeled complex or the labeledmarker-analogue by immune reaction or other reaction in the detectionzone, thus building up a visible test line in the detection zone duringthe process. Preferably, the label is an optically detectable label.Forming a complex at the test line concentrates and immobilizes thelabel and the test line becomes visible to the naked eye, indicating apositive test result. Particularly preferred are direct labels, and moreparticularly gold labels which can be best recognized by the naked eye.Additionally, an electronic read out device (e.g. on the basis of aphotometrical, acoustic, impedimetrical, potentiometric and/oramperometric transducer) can be used to obtain more precise results anda semi-quantification of the analyte. Other labels may be latex,fluorophores, or phosphorophores.

Furthermore, this invention includes a device and test kit for theperformance of the described method.

In some preferred embodiments, the specific binding partners for theanalytes in the sample are monoclonal, polyclonal, single domain orrecombinant antibodies, or fragments of antibodies capable of binding toa pathogen. Alternatively, the specific binding partners may also beantigens capable of binding to antibodies against a pathogen or anallergen. Other types of binding partners include, but are not limitedto, bioorganic macromolecules like aptamers or ligands/receptors,nanoparticles, or nucleic acids.

The visual label may be any label visible to the naked eye, including,but not limited to, colored particles such as colloidal gold, dyed latexbeads, selenium, or carbon. In some embodiments, the visual tags arealso coated with fluorescing elements. In some embodiments, thefluorescing element is a fluorescing dye. Alternatively, a mixture ofpreferably colorless fluorescing latex bead conjugates are mixed withcolloidal gold (a visible spectrum) conjugates, or conjugates producinga visible read test line, in lateral flow assays to enhance sensitivityof the assay and to aid in visually reading true positives and truenegatives. In embodiments where nanoparticles are used, thenanoparticles that may be used include, but are not limited to,selenium, carbon, and colloidal gold.

Preferred targets include, but are not limited to, proteins,glycoproteins, proteoglycans, nucleic acids, and lipoproteins. Otherpreferred targets include, but are not limited to, pathogens,low-molecular-weight compounds, and/or allergy-associated components.The pathogens are preferably selected from viruses, microorganisms, e.g.bacteria, and parasites, e.g. amoebae or nematodes. Theallergy-associated components are preferably selected from allergens andanti-allergic components.

In some preferred embodiments, the sample is a sample of body fluid. Inthese embodiments, the sample of body fluid is preferably taken from abody surface selected from mucosal membrane fluids (preferably of theoral, nasal, vaginal, and ocular cavities), blood, urine, tears,cerebrospinal fluid, secretions from glands and secretions from lesionsor blisters, e.g. lesions or blisters on the skin. More preferably, thesample is selected from oral, nasal, ocular, genital and rectal fluid,secretions from skin lesions or blisters, CSF (cerebral spinal fluid),and exudates. In some embodiments, the body fluid samples are preferablyfluids that do not flow once collected.

In embodiments where mucosal membrane fluids are used, at least onemucolytic agent is preferably used to breakdown the mucus and make thetarget more accessible for testing.

Lateral flow devices are known, and are described in, e.g., U.S.Published Patent Application Nos. 2005/0175992 and 2007/0059682. Thecontents of both of these applications are incorporated herein byreference. Other lateral flow devices known in the art couldalternatively be used with the systems and methods of the presentinvention.

U.S. Published Patent Application No. 2007/0059682, discloses detectingan analyte and a sample which can also contain one or more interferingsubstances. This publication teaches separating the analyte from theinterfering substances by capturing the interfering substances on thechromatographic carrier, and detecting the analyte on the carrierseparated from the interfering substances.

U.S. Published Patent Application No. 2005/0175992 discloses a methodfor detecting targets, such as pathogens and/or allergy-associatedcomponents, in a human body fluid where the body fluid sample iscollected by a collection device, such as a swab member. The samples aretransferred from the swab member to a sample analysis device, on whichan analysis of the targets can occur by immunochemical or enzymaticmeans. The test result is capable of being displayed within a very shortperiod of time and can be directly read out by the user. This enablespoint-of-care testing with results available during a patient visit. Theinventions disclosed in this application are particularly advantageousfor the diagnosis of conjunctivitis.

The chromatographic test strip shown in FIGS. 1 through 4 includes aplurality of different strip materials. The device preferably includesan absorbent pad 1, an application zone 2, a detection zone 3, and awaste zone 4. The strip materials are arranged on an adhesive plasticbacking 5. The absorbent pad 1 is provided in this example for adding anelution medium in order to facilitate the transfer of the sample to thedetection zone 3. US Published Patent Application No. 2007/0059682,describes methods to increase specificity of lateral flow immunoassays.These methods could also be used in combination with the embodimentsdescribed herein.

FIG. 2 shows a housing 6, which is preferably plastic, containing thestrip as shown in FIG. 1. A sample application window 7 brings acollection device into contact with the strip. The test result isdisplayed in the read out window 8. FIG. 3 shows the collection devicefor collecting a sample. In one example, the collection device is a swabmember. The collection device includes a body 9, which is preferablyplastic, with a sample collection material 11 fixed on it and an opening10 corresponding to a read out window when the collection device isoperatively in contact with a test strip. FIG. 4 shows a test kit, whichincludes the sample analysis device of FIGS. 1 and 2 and the collectiondevice of FIG. 3.

The methods and devices of the present invention incorporate a lysiszone including at least one lysis agent, and/or a mucolysis zoneincluding at least one mucolytic agent, as part of a lateral flow teststrip, such as those shown in FIGS. 1 through 4, or other lateral flowdevices known in the art, in order to break down the sample material insitu.

The present invention is suitable for various methods for loading thesample. The assay will either be started directly when sample istransferred in a sufficient volume of liquid, such as a body fluid, orthe process may require that a sample be added to or eluted by a sampletransport liquid (e.g. tap water or a buffer solution). In one preferredembodiment, a sample which has been collected, such as by a swab, istransferred directly onto the sample application zone of a test strip.In this embodiment, a sample transport liquid is then added to the teststrip. In another preferred embodiment, a liquid sample is depositeddirectly onto the sample application zone of a test strip. In thisembodiment, the liquid sample itself, if of sufficient volume, becomesthe transport liquid. If the volume of the liquid sample isinsufficient, then a sample transport liquid is additionally added. Inyet another preferred embodiment, a liquid sample is pre-mixed with thesample transport liquid and then both are applied to the test striptogether.

Following sample loading, sample traveling with the transport liquidwill encounter one or more lysis agents and/or one or more mucolyticagents. The lysis or mucolytic agent agent will have been pre-loadedonto the test strip and is eluted by the transport liquid. In somepreferred embodiments the lysis or mucolytic agent has been dried intothe test strip. Alternatively, the lysis or mucolytic agent may bepre-dried by freeze drying or lyophilizing and then pre-loaded into thetest strip. In other embodiments, the lysis agent or the mucolytic agentmay be absorbed, adsorbed, embedded or trapped on the test strip. In apreferred embodiment, the lysis agent or the mucolytic agent islocalized between the sample application zone and the conjugate zone.The lysis agent or the mucolytic agent is preferably soluble or misciblein the sample transport liquid, and the lysis agent or mucolytic agentis solubilized and activated upon contact with the sample transportliquid. The sample transport liquid then contains both lysis and/ormucolytic agent (in solution or suspension) and sample components (insuspension). Any lysis-susceptible or mucolysis-susceptible componentsin the sample, then being exposed in suspension to the lysis agentand/or the mucolytic agent, are themselves lysed or mucolysed in situ.The running buffer then carries the analyte, including any lysis ormucolysis-freed components, through the conjugate zone and to thedetection zone.

The location where the lysis agent and/or the mucolytic agent ispre-loaded can be varied as needed. In order to maximize the time thatthe sample has to interact with the lysis or mucolytic agent as well asto minimize the amount of lysis or mucolytic agent reaching thedetection zone, the dried, absorbed, adsorbed, embedded, or trappedlysis or mucolytic agent may be located in or just downstream of thesample application zone. Or, in order to minimize the distance in whichthe lysed or mucolysed product must travel before reaching the conjugatezone, the lysis or mucolytic agent may be located closer to theconjugate zone.

In some preferred embodiments, more than one lysis agent, more than onemucolytic agent, or a combination of one or more lysis agents and one ormore mucolytic agents may be used.

The concentration of lysis agent pre-loaded onto a test strip ispreferably between 0.001% and 5% weight/volume. The volume to bepre-loaded depends on where the lysis agent is pre-loaded. Appropriateranges are 1 to 10 microliters when pre-loaded into the sample collectorfleece (the sample application zone) or 5 to 50 microliters whenpre-loaded into the absorbent pad or into other locations within thetest strip. Ideally, the amount pre-loaded should be approximately 3microliters pre-loaded into the sample collector fleece or approximately10 microliters pre-loaded into the absorbent pad or into other locationswithin the test strip.

The concentration of mucolytic agents pre-loaded onto a test strip ispreferably between 0.01% and 10% weight/volume (or between 0.01% and 10%volume/volume). Similar to the discussion above with respect to lysisagents, the volume of the mucolytic agent to be pre-loaded depends onwhere the mucolytic agent is pre-loaded.

Selection of a specific lysing environment and agent will depend on theanalyte and the assay. pH and ionic strength are key to the lysingenvironment. As to pH established by the lysis agent, a pH below 4.0tends to precipitate materials, especially proteins. Higher pH, aboveapproximately 10.0, tends to lyse materials such as proteins and cellswalls. Therefore, a pH of approximately 10.0 or above is preferable formany applications. Alternatively, lower pH may be preferred for nucleicacid targets. Similar considerations can be considered when selecting aspecific mucolytic environment.

As to ionic strength established by the lysis agent, both high and lowionic strength may be used to lyse. For example, a lower ionic strength(hypotonic) tends to break up erythrocytes. Water by itself can lyseerythrocytes. Higher ionic strength environments may be used to rupturecertain cell walls and membranes.

As to specific lysis agents, they may be grouped and selected based ontheir properties: salts, amphoteric and cationic agents, ionic andnon-ionic detergents. The salt, Ammonium Chloride (NH₄Cl), lyseserythrocytes. Other salts, including, but not limited to, highconcentrations of Sodium Chloride (NaCl) and Potassium Chloride (KCl),may rupture certain cell walls and membranes. Other lysis agents areamphoteric agents including, but not limited to, Lyso PC, CHAPS, andZwittergent. Alternatively, cationic agents including, but not limitedto, C16 TAB and Benzalkonium Chloride may be used as a lysis agent. Bothionic and non-ionic detergents are often used to break or lyse the cellwall or cell membrane components such as lipoproteins and glycoproteins.Common ionic detergents include, but are not limited to, SDS, Cholate,and Deoxycholate. Ionic detergents are good solubilizing agents.Antibodies retain their activity in 0.1% SDS or less. Common non-ionicdetergents include, but are not limited to, Octylglucoside, Digitonin,C12E8, Lubrol, Triton X-100, Noniodet P-40, Tween 20, and Tween 80.Non-ionic and mild ionic detergents are weaker denaturants and often areused to solubilize membrane proteins such as viral surface proteins.Additional lysis agents include, but are not limited to, urea andenzymes. Combinations of different lysis agents may be used to optimizethe lysing environment.

Surfactants are generally wetting agents and lower the surface tensionof a liquid. This then allows easier spreading by lowering theinterfacial tension between liquids. So, surfactants can interfere withthe natural binding of antigen and antibody or ligand and receptors. Theconcentrations are, therefore, experimentally chosen for each class oflysis agent. Once lysis occurs, it is important that the desired bindingreactions not be hindered. Generally, 0.001% lysis agent concentrationis considered the lower limit, and the upper limit is approximately 1%.There is an additive or synergistic effect when combinations of lysisagents are used. This expands the working range of concentration to runfrom approximately 0.001% to 1%. Finally, some undesirable non-specificbinding may be prevented at a Tween 20 concentration of 5%. In allcases, the total amount of lysis agent pre-loaded onto all locations ofan individual test strip must be sufficient to lyse barriers toimmunodetection, permitting practical operation of the test strip.

Any mucolytic agent capable of effectively degrading the mucus in thesample could be used. Some examples for mucolytic agents includeproteolytic enzymes including, but not limited to, Lysozyme, Trypsin andChymotrypsin. Lipases and Nucleases can also be used alone or incombination. In certain applications, chaotropic agents and chelatingagents such as EDTA can also be used. Some specific examples include,but are not limited to, N-acetyl cysteine (NAC, aka N-acetyl-LCysteine), Acetylcysteine, Ambroxol, Bromhexine, Carbocisteine,Domiodol, Dornase alpha, Eprazinone, Erdosteine, Guiafenesin Letosteine,Mesna (2-MercaptoEthane Sulfonate sodium), Neltenexine, Sobrerol,Stepronin, and Tiopronin.

In a preferred embodiment, NAC is used. NAC has many properties thatmake it an ideal mucolytic agent for in situ mucolysis in point of careassays. NAC is soluble in both aqueous and non-aqueous solutions. Thesolubility and the toxicity of NAC are irrelevant at the levels neededfor mucolysis on chromatographic test strips. In a preferred embodiment,the amount of NAC in the solution ranges from approximately 0.01 to 10%of the total volume (weight/volume or volume/volume) of the solution.NAC is known to be bio-compatible so it can be used even on samplecollection materials that come in direct contact with bodily fluids. NACcannot be immobilized on the nitrocellulose since it is not a proteinbut is a modified amino acid. The SH group on NAC confers the mucolyticactivity of NAC and the SH group on NAC is the preferred moiety forpassive conjugation to colloidal gold. The SH group on NAC also makes itsuitable for “directional” chemical binding to microspheres such aslatex beads. NAC is stable in dried form as well as in buffers. NACshould be thermally stable or unaffected by Ethylene Oxide (EtO) andhence, can remain active on a test strip after sterilization of the teststrip with NAC on it.

In another preferred embodiment, a modified form of Acetyl cysteinecould be used as the mucolytic agent. In its modified form, the Acetylgroup goes on the Cysteine moiety not through the NH₂ end as in n-AcetylCysteine but goes on to the Cysteine moiety through the Beta Carbon'sCH₃ group.

The lysis agent or mucolytic agent itself preferably does not interferewith any other assay detector or indicator agents and thus does notinterfere with any other assay interactions and reactions to such anextent as to prevent practical operation of the assay. A lysis agent ormucolytic agent should have sufficient shelf life to allow manufacture,distribution and storage before use of a test strip in point-of-caretesting.

In a preferred embodiment of the present invention, the lateral flowassay device of the present invention includes a sample-transportingliquid, which can be a buffer, and a chromatography test stripcontaining one or several fleece materials or membranes with capillaryproperties through which sample flows. In a device and method of theinvention, it is unnecessary to lyse the cells in the sample prior toapplying it to the test strip.

In a preferred embodiment, as shown in FIGS. 5A through 5D, the sampleis applied to the application zone 201 on a chromatography test strip200. The sample passes a lysis zone 250, where a lysis agent will havepreferably been pre-loaded onto the test strip and is eluted by thetransport liquid. The lysis agent lyses any lysis-susceptible componentsin the sample in situ.

The chromatographic test strip contains a sample application zone 201, alysis zone 250 containing a lysis agent, and a conjugate zone 260containing at least one labeled binding partner that is eluted by andthen able to migrate with a sample transport liquid (e.g. a buffersolution). The labeled binding partner is capable of specificallybinding to an analyte of interest to form a conjugate which in turn iscapable of specifically binding to another specific reagent or bindingpartner in the detection zone. Although not shown in these Figures, anabsorbent pad, similar to the absorbent pad 1 shown in FIG. 1, as wellas other known lateral flow assay components including, but not limitedto, a waste zone, a carrier backing, a housing and an opening in thehousing for result read out may optionally also be a component of thetest strip 200 in this embodiment.

In a preferred embodiment, the lysis agent is localized in the lysiszone 250 between the sample application zone 201 and the conjugate zone260. The lysis agent is preferably soluble or miscible in the sampletransport liquid, and the lysis agent is solubilized and activated uponcontact with the sample transport liquid. The sample transport liquidthen contains both lysis agent in solution or suspension and samplecomponents in suspension. Any lysis-susceptible components in a sample,then being exposed in suspension to the lysis agent, are themselveslysed in situ. The running buffer then carries the analyte, includingany lysis-freed components, to the detection zone 205.

The lysis zone 250 is preferably located between the sample applicationzone 201 and the conjugate zone 260, as shown in FIG. 5A. In otherembodiments, the lysis zone 250 overlaps the sample application zone201, the conjugate zone 260 or both the sample application zone 201 andthe conjugate zone 260 as shown in FIGS. 5B, 5C, and 5D, respectively.Note that the figures are schematic, and are not drawn to scale. Theamount of overlap between the different zones (as shown in FIGS. 5Bthrough 5D) may be highly variable.

The test strip 200 also includes a detection zone 205 containing a firstsection for detection of a first analyte, e.g. a test line 202,including an immobilized specific binding partner, complimentary to theconjugate formed in and arriving from the conjugate zone (260). Thus, atthe test line 202, detection zone binding partners trap the labeledbinding partners from the conjugate zone 260 along with their boundanalytes. This localization of the analytes with their labeled bindingpartners gives rise to an indication at the test line 202. At the testline 202, the presence of an analyte is determined by qualitative and/orquantitative readout of the test line 202 indication resulting from theaccumulation of labeled binding partners. Optionally, the detection zone205 may contain further test lines to detect other analytes, as well asa control line 204. The control line 204 indicates that the labeledspecific binding partner traveled through the length of the assay, eventhough it may not have bound any analyte, thus confirming properoperation of the assay. As shown in FIGS. 5A through 5D, the controlzone 204 is preferably downstream of the test zone 202. However, inother embodiments, the control zone 204 may be located upstream of thetest zone 202.

In a preferred embodiment, the control line 204 includes an antibody orother recombinant protein which binds to a component of the elutionmedium or other composition being used in the test. In embodiments wherenucleic acids are the targets, the control line (204) preferablyincludes a nucleic acid complementary to the labeled nucleic acid beingused as a binding partner for the target nucleic acid.

Although only one test line is shown in the figures, multiple test linesare within the spirit of the invention. In some embodiments where thereare multiple targets, the presence of each target preferably correspondsto a separate test line 202. In other embodiments where there aremultiple targets, the presence of multiple targets may be indicated onthe same test line such that the presence of more than one target hasdifferent characteristics than the presence of a single target. Forexample, the presence of multiple targets on the same test line may bevisually indicated by a different color than the presence of each of thetargets alone.

In other embodiments, it is possible to have one or more mild lysisagents in the running buffer itself. In these embodiments, there is noadverse effect on the conjugate zone which will be downstream and thesample can either be upstream or downstream of the conjugate zone. Alysing enzyme in the running buffer can “target” its substrate and cutit to open up the cell wall. As an example, penicillin can excise or“punch a hole” in a susceptible bacteria. In other embodiments, when thelysis agent is applied to the sample collection material 11 (see FIG.3), then the conjugate zone may be upstream of the sample applicationzone.

In another preferred embodiment, a barrier may be disposed in a“blocking zone” between the sample application zone and either theconjugate zone or the detection zone, preferably before the conjugatezone. In this case, the lysis agent is pre-loaded in the sampleapplication zone or between the sample application zone and the barrier.Thus, lysis occurs before the sample reaches the barrier, and thebarrier serves to slow or arrest those lysed materials effectivelylarger than the porosity of the barrier while permitting effectivelysmaller materials to pass more easily. Thus, the barrier provides afiltering effect and reduces interference with binding interactions inthe conjugate and detection zones. Selection of a specific barriermaterial depends on the analyte and the assay.

A blocking zone barrier may be physical or biological. Examples ofphysical barriers include glass fiber matrices which inherently bind ortrap erythrocytes and their cellular debris. Other physical matrices maybe “sieve-type” matrices, as in a filtering system, where the small poresize blocks passage of cells but does allow passage of biomarkers.

In contrast, biological barriers are immobilized biological materialsthat specifically bind to ligands or receptors on a cell surface,preventing the cells from flowing further. Examples include antibodies,recombinant proteins, specific lectins, and receptors/ligands.Biological materials may also be combined into physical barriers such asglass fiber membranes.

FIGS. 6A through 6D show a barrier disposed in a “blocking zone” 170between the sample application zone 101 and either the conjugate zone160 (FIGS. 6A and 6C) or the detection zone 105 (FIGS. 6B and 6D) of thetest strip 100. In either case, the lysis zone 150 either overlaps withthe sample application zone 101 such that the lysis agent is pre-loadedin the sample application zone 101 (see FIGS. 6C and 6D) or the lysiszone 150 is located between the sample application zone 101 and thebarrier in the blocking zone 170 such that the lysis agent is pre-loadedbetween the sample application zone 101 and the blocking zone 170 (seeFIGS. 6A and 6B). Thus, lysis occurs before the sample reaches theblocking zone 170, and the barrier in the blocking zone 170 serves toslow or arrest those lysed materials effectively larger than theporosity of the barrier while permitting effectively smaller materialsto pass more easily. Thus, the barrier provides a filtering effect andreduces interference with binding interactions in the conjugate 160 anddetection zones 105. Selection of a specific barrier material depends onthe analyte and the assay. Similar to FIGS. 5A through 5D, the detectionzone 105 includes at least one test zone 102 and a control zone 104.

In another preferred embodiment, shown in FIGS. 7A through 7D, thesample is applied to the application zone 301 on a chromatography teststrip 300. The sample passes a mucolytic zone 350, where one or moremucolytic agents have preferably been pre-loaded onto the test strip,and is eluted by the transport liquid. The mucolytic agent breaks down amucosal sample in situ.

The chromatographic test strip contains a sample application zone 301, amucolytic zone 350 containing a mucolytic agent, and a conjugate zone360 containing at least one labeled binding partner that is eluted byand then able to migrate with a sample transport liquid (e.g. a buffersolution). The labeled binding partner is capable of specificallybinding to an analyte of interest to form a conjugate which in turn iscapable of specifically binding to another specific reagent or bindingpartner in the detection zone. Although not shown in these Figures, anabsorbent pad, similar to the absorbent pad 1 shown in FIG. 1, as wellas other known lateral flow assay components including, but not limitedto, a waste zone, a carrier backing, a housing and an opening in thehousing for result read out may optionally also be a component of thetest strip 300 in this embodiment.

In a preferred embodiment, the mucolytic agent is localized in themucolytic zone 350 between the sample application zone 301 and theconjugate zone 360. The mucolytic agent is preferably soluble ormiscible in the sample transport liquid, and the mucolytic agent issolubilized and activated upon contact with the sample transport liquid.The sample transport liquid then contains both mucolytic agent insolution or suspension and sample components in suspension. Anymucolysis-susceptible components in a sample, then being exposed insuspension to the mucolytic agent, are broken down in situ. The runningbuffer then carries the analyte, including any mucolysis-freedcomponents, to the detection zone 305.

The mucolytic zone 350 is preferably located between the sampleapplication zone 301 and the conjugate zone 360, as shown in FIG. 7A. Inother embodiments, the mucolytic zone 350 overlaps the sampleapplication zone 301, the conjugate zone 360 or both the sampleapplication zone 301 and the conjugate zone 360 as shown in FIGS. 7B,7C, and 7D, respectively. Note that the figures are schematic, and arenot drawn to scale. The amount of overlap between the different zones(as shown in FIGS. 7B through 7D) may be highly variable.

The test strip 300 also includes a detection zone 305 containing a firstsection for detection of a first analyte, e.g. a test line 302,including an immobilized specific binding partner, complimentary to theconjugate formed in and arriving from the conjugate zone 360. Thus, atthe test line 302, detection zone binding partners trap the labeledbinding partners from the conjugate zone 360 along with their boundanalytes. This localization of the analytes with their labeled bindingpartners gives rise to an indication at the test line 302. At the testline 302, the presence of an analyte is determined by qualitative and/orquantitative readout of the test line 302 indication resulting from theaccumulation of labeled binding partners. Optionally, the detection zone305 may contain further test lines to detect other analytes, as well asa control line/zone 304. The control line 304 indicates that the labeledspecific binding partner traveled through the length of the assay, eventhough it may not have bound any analyte, thus confirming properoperation of the assay. As shown in FIGS. 7A through 7D, the controlline 304 is preferably downstream of the test zone 302. However, inother embodiments, the control line 304 may be located upstream of thetest zone 302.

In a preferred embodiment, the control line 304 includes an antibody orother recombinant protein which binds to a component of the elutionmedium or other composition being used in the test. In embodiments wherenucleic acids are the targets, the control line 304 preferably includesa nucleic acid complementary to the labeled nucleic acid being used as abinding partner for the target nucleic acid.

Although only one test line is shown in the figures, multiple test linesare within the spirit of the invention. In some embodiments where thereare multiple targets, the presence of each target preferably correspondsto a separate test line 302. In other embodiments where there aremultiple targets, the presence of multiple targets may be indicated onthe same test line such that the presence of more than one target hasdifferent characteristics than the presence of a single target. Forexample, the presence of multiple targets on the same test line may bevisually indicated by a different color than the presence of each of thetargets alone.

In other embodiments, it is possible to have one or more mucolyticagents in the running buffer itself. In these embodiments, there is noadverse effect on the conjugate zone which is downstream of where thebuffer is added and the sample can either be upstream or downstream ofthe conjugate zone. A mucolytic agent in the running buffer can “target”the mucosal sample and break it up. In other embodiments, when themucolytic agent is applied to the sample collection material 11 (seeFIG. 3), then the conjugate zone may be upstream of the sampleapplication zone.

In other embodiments, the sample collector, for example a swab member,can be treated with the mucolytic agent, as long as the agent isbio-compatible. For example, a swab member can be treated with NAC suchthat NAC is dried onto the swab before the sample is collected. Even ifthe NAC leaks into the body, for example into the eyes or otherorifices, there is no harm because it is bio-compatible. Here, a samplewith a mucous type membrane would be “clarified” as the mucous layer isbroken down. This is particularly useful in samples used to test forsexually transmitted infections, for example, with organisms likeChlamydia. If NAC is used, there is no need to use one swab (or cottonball) to “clean” the environment before the second swab collects thesample that will be tested for Chlamydia.

In another preferred embodiment, a barrier may be disposed in a“blocking zone” between the sample application zone and either theconjugate zone or the detection zone, preferably before the conjugatezone. In this case, the mucolytic agent is pre-loaded in the sampleapplication zone or between the sample application zone and the barrier.Thus, mucolysis occurs before the sample reaches the barrier, and thebarrier serves to slow or arrest any broken down materials effectivelylarger than the porosity of the barrier while permitting effectivelysmaller materials to pass more easily. Thus, the barrier provides afiltering effect and reduces interference with binding interactions inthe conjugate and detection zones. Selection of a specific barriermaterial depends on the analyte and the assay.

A blocking zone barrier may be physical or biological. Examples ofphysical barriers include glass fiber matrices which inherently bind ortrap erythrocytes and their cellular debris. Other physical matrices maybe “sieve-type” matrices, as in a filtering system, where the small poresize blocks passage of cells but does allow passage of biomarkers.

In contrast, biological barriers are immobilized biological materialsthat specifically bind to ligands or receptors on a cell surface,preventing the cells from flowing further. Examples include antibodies,recombinant proteins, specific lectins, and receptors/ligands.Biological materials may also be combined into physical barriers such asglass fiber membranes.

FIGS. 8A through 8D show a barrier disposed in a “blocking zone” 470between the sample application zone 401 and either the conjugate zone460 (FIGS. 8A and 8C) or the detection zone 405 (FIGS. 8B and 8D) of thetest strip 400. In either case, the mucolytic zone 450 either overlapswith the sample application zone 401 such that the mucolytic agent ispre-loaded in the sample application zone 401 (see FIGS. 8C and 8D) orthe mucolytic zone 450 is located between the sample application zone401 and the barrier in the blocking zone 470 such that the mucolyticagent is pre-loaded between the sample application zone 401 and theblocking zone 470 (see FIGS. 8A and 8B). Thus, mucolysis occurs beforethe sample reaches the blocking zone 470, and the barrier in theblocking zone 470 serves to slow or arrest those materials effectivelylarger than the porosity of the barrier while permitting effectivelysmaller materials to pass more easily. Thus, the barrier provides afiltering effect and reduces interference with binding interactions inthe conjugate 460 and detection zones 405. Selection of a specificbarrier material depends on the analyte and the assay. Similar to FIGS.7A through 7D, the detection zone 405 includes at least one test zone402 and a control zone 404.

The mucolytic agents may alternatively be upstream of the sampleapplication zone (or overlapping the sample application zone on theupstream end of the strip), for example in the absorbent pad 1 shown inFIG. 1. In other embodiments, there may be multiple mucolytic agentsand/or lysis agents in more than one location on the strip relative tothe other components on the test strip, or in the running buffer.

In some preferred embodiments of the present invention, the lateral flowdevice of the present invention includes a sample-transporting liquid,which can be a buffer, a sample compressor, and a chromatography teststrip containing one or several fleece materials or membranes withcapillary properties through which sample flows. In a device and methodof the invention, it is unnecessary to break down the cells in thesample prior to applying the sample to the test strip.

FIG. 9 shows a sample analysis device (test strip) 901 and a samplecollector 902. The sample collector 902 may be any type of samplecollector 902 known in the art, for example the sample collector 902could be a swab member. The sample 920 may include the analyte 903, aswell as interfering particles 905 (which may include interferingproteins or interfering genes) and other interfering particles or celldebris 904. The sample analysis device 901 includes a conjugate zone 908upstream of the sample application zone 918 in this figure. Although theconjugate zone 908 is shown upstream of the sample application zone 918in this figure, the conjugate zone 908 may alternatively overlap thesample application zone 918 or be downstream of the sample applicationzone 918 within the spirit of the present invention. The sampleapplication zone 918 is also a microfiltration zone, which preferablyfilters out cell debris and interfering particles 904 that are in thesample 920.

The conjugate zone 908 preferably includes both a mobile conjugate 915,which includes a portion that binds to the analyte 903 and a detectablelabel, and a control zone binding partner 916 with a detectable label,which may be, for example, a control zone antibody with a visual label.In some embodiments, the mobile conjugate is a test antibody conjugatewith a visual label. The control zone binding partner 916 binds with animmobilized binding partner for it in the control zone 911 and indicateswhether the test has run correctly. If the analyte 903 is present in thesample 920, the analyte binds to the conjugate 915, and the conjugate915-analyte 903 complex travel to the test zone 910 in the detectionzone 912. The analyte 903 then binds to an immobilized binding partner917 for the analyte 903, to form the full “sandwich” in a sandwich-typeassay.

The transfer of the sample from the sample collector 902 to the sampleapplication zone 918 on the sample analysis device is preferably adirect transfer, i.e. the transfer takes place without pretreatment ofthe sample on the sample collector 902. In embodiments withoutpretreatment of the sample or the sample collector 902, pressure 914 isapplied and microfiltration occurs in the region where the samplecollector fleece directly contacts the fleece on the sample analysisdevice 901. The fibers of the fleece interlock to form a grating orphysical interference. Thus, larger elements contained in the sample,for example cell debris and interfering particles 904 are held back andnot eluted.

The sample application device 901 preferably also includes a blockingzone 909 that includes one or more capturing reagents. This blockingzone captures interfering proteins and/or genes 905 that may be in thesample 920. Capture of an interfering substance 904, 905 by one or morecapturing reagents occurs when the capturing reagent interacts in somemanner with the interfering substance to keep the interfering substancefrom interfering with the detection of the analyte. While a blockingzone 909 is shown in FIG. 9, the capturing reagents may be located in acapturing zone 909 made of materials that allow the capturing reagentsto be mobile, in the elution medium, mixed and dried with the reagents,incorporated into the sample application zone, incorporated into thesample collector fleece material, and/or immobilized on an immobilizingmaterial (for example, nitrocellulose) either as a line or a zone. Anyof these or any combination of these may be used in the embodiments ofthe present invention, depending on the test and sample matrix.

The sample analysis device 901 also optionally includes an absorbent pad907 upstream of the conjugate zone 908 and the sample application zone918. Buffer is added and travels in the direction of the arrow 906 toelute the test components, including the sample 920, the conjugate 915,and the control zone binding partner 916, to the detection zone 912. Thesample analysis device 901 also preferably includes a waste pad 913 atthe downstream end of the device 901. The sample analysis device 901 mayalso optionally include a backing 923.

One or more lysis or mucolytic agents may be located (for example, driedon or otherwise included in the membranes making up these zones) in anyof various locations on the test strip 901, including within theabsorbent pad 907, the conjugate zone 908, the sample application zone918 or the blocking zone 909, or overlapping any of the intersectionsbetween these areas on the strip. In other embodiments, the lysis ormucolytic agents may be on the sample collector 902 as long as the lysisor mucolytic agent being used is biocompatible. In yet anotherembodiment, one or more lysis or mucolytic agents may be placed wherethe sample application zone 918 meets the nitrocellulose membrane (useof a nitrocellulose membrane on the strip preferably begins in theblocking zone 909 in this figure; however, if there may alternatively beno blocking zone 909 on the strip). In preferred embodiments, the lysisor mucolytic agent overlaps or is upstream of the sample applicationzone 918. In still other embodiments, one or more lysis or mucolyticagent may be in the running buffer. If there are multiple lysis and/ormucolytic agents, they can be located in the same or different locationson the test strip or on the sample collector. A single lysis and/ormucolytic agent could be located in more than one location, if desired.

In some embodiments, the devices and methods of the present inventioninclude a sample compressor 1030. Some schematic examples of samplecompressors 1030 that could be used are shown in FIGS. 10A and 10B. Thesample compressors 1030 preferably include a handle 1031, an extendedportion 1032, and a pad portion 1033. In some designs, the samplecompressor includes additional sections, such as a ledge portion 1034that the pad portion 1033 is placed upon. While specific examples areshown in FIGS. 10A and 10B, any sample compressor 1030 that is able toexert pressure to transfer one or more components of the assay and thesample to the sample analysis device could be used in the embodiments ofthe present invention. In preferred embodiments, the conjugate 1036(FIG. 11C) is pre-loaded and dried onto a pad 1033 that forms theconjugate zone. In some preferred embodiments, a labeled control 1061(FIG. 11C) that is able to complex with a binding partner at the controlzone is also pre-loaded and dried onto the pad 1033 of the samplecompressor 1030. In other preferred embodiments, the second bindingpartner 1038 (FIG. 13C) for the analyte is located on the pad 1033. Insome preferred embodiments, at least one lysis or mucolytic agent ispre-loaded and dried onto the pad 1033 that forms the conjugate zone.Any combination of the conjugate 1036, the second binding partner 1038,one or more lysis and/or mucolytic agents, or the control zone bindingpartner 1061 may be on the pad portion 1033 of the sample compressor1030.

FIG. 10C shows an example of a sample collector 1035. In this example,the sample collector 1035 is a swab member. The sample collector 1035preferably includes a sample collection portion 1060, which ispreferably made of fleece-type materials. In some embodiments, thesample collector 1035 is sterile. In some embodiments, one or more lysisor mucolytic agents that are biocompatible may be pre-loaded and driedonto the collection portion 1060 of the sample collector 1035. In onepreferred embodiment, NAC is pre-loaded and dried onto the collectorportion 1060 of the sample collector 1035.

FIGS. 11A through 11C show one embodiment of a system with a samplecompressor 1030, a sample collector 1035, and a sample analysis device(a test strip in the figure). The test strip preferably includes anabsorbent pad 1042, a sample application zone 1044, a detection zone1052, and an optional waste pad 1047. The test strip also preferablyincludes a carrier backing 1048. The detection zone 1052 preferablyincludes a test zone 1045, which includes an immobilized binding partner1038 for the analyte 1040, as well as a control zone 1046. In thisembodiment, the conjugate 1036 is on the sample compressor 1030. Thefirst binding partner 1037, which is part of the conjugate 1036, fromthe sample compressor 1030 binds the analyte 1040 in the test sample toform a half sandwich, which is then transported to the second bindingpartner 1038 which is immobilized in a test zone 1045. The full sandwich1120 that forms between the portion 1037 of the conjugate 1036 thatbinds to the analyte 1040, the analyte 1040, and the second bindingpartner 1038 is shown in FIG. 11B. In preferred embodiments, the pad1033 on the sample compressor 1030 also includes a control zone bindingpartner 1061 with a detectable label. The control zone binding partner1061 complexes with its binding partner in the control zone 1046.Including the control zone binding partner 1061 on the sample compressor1030, instead of on the test strip or in the buffer as known in theprior art, permits the user to be sure that the components on the samplecompressor 1030, which, in this embodiment include both the conjugate1036 and the control zone binding partner 1061, have effectivelytransferred to the sample analysis device and thus ensures properoperation of the system.

In one example, both the first binding partner 1037 and the secondbinding partner 1038 are different antibodies to the analyte. Thecontrol zone binding partner 1061 is also preferably an antibody, andits binding partner at the control zone is an antigen (or vice versa).In other embodiments, specific binding partners may also be antigenscapable of binding to antibodies against the analyte. Other types ofbinding partners are bioorganic macromolecules like aptamers orreceptors, nanoparticles, or nucleic acids. The device shown in FIGS.11A-11C of the present invention can be used for any binding assays, andcan avoid the use of antibody/antigens or nucleic acids, for example, inligand-receptor binding assays and enzyme-substrate binding assays.

In operation, the sample collector 1035 is placed such that the sampleis directly above the sample application zone 1044. In some embodiments,placement of the sample collector 1035 above the sample application zone1044 is not simultaneous with placement of the sample compressor 1030.In other words, in these embodiments, some of the sample is transferredto the sample application zone 1044 before the sample compressor 1030 isadded to the vertical stack.

The sample compressor 1030 exerts pressure 1051 on the sample collector1035, using pressure to transfer the sample, including the analyte 1040(if present), and the conjugate 1036 onto the sample application zone1044. If there is also a control zone binding partner 1061 on the samplecompressor 1030, the control zone binding partner 1061 is alsotransferred. Note that the transfer is due to pressure, not due to flowor capillary action. Then, buffer 1043 is added to permit flow of theconjugate 1036-analyte 1040 complex (if present) to the detection zone1052. An immobilized binding partner 1038 in the test zone 1045 thenbinds the analyte, forming the complete sandwich. Since the conjugate1036 includes a label 1041, the complex that forms is detectable andindicates a positive result. Proper operation of the test also resultsin a detectable positive result in the control zone 1046 due to theinteraction between the control zone binding partner 1061 and itsimmobilized partner in the control zone 1046.

One or more lysis or mucolytic agents may be located (for example, driedon or otherwise included in the membranes making up these zones) on thetest strip in various locations including, in the absorbent pad 1042, inthe sample application zone 1044, directly upstream of the detectionzone 1052, in the detection zone itself, or within any of theintersections between these sections of the test strip. Alternatively,one or more lysis or mucolytic agents is pre-loaded and dried on thesample compressor 1030. One or more lysis or mucolytic agents mayalternatively be located in the buffer. In other embodiments, the lysisor mucolytic agents may be on the sample collector as long as the lysisor mucolytic agent being used is biocompatible. In yet anotherembodiment, one or more lysis or mucolytic agents may be placed wherethe sample application zone 1044 meets the detection zone 1052 or wherethe sample application zone meets the nitrocellulose membrane. Inpreferred embodiments, the lysis or mucolytic agent overlaps or isupstream of the sample application zone 1044 or is located on the samplecompressor 1030. If there are multiple lysis and/or mucolytic agents,they can be located in the same or different locations on the teststrip, on the sample collector, or on the sample compressor. A singlelysis and/or mucolytic agent could be located in more than one location,if desired.

In other embodiments, the conjugate zone contains both the bindingpartners for the analyte in the sample to form a “full sandwich”. One ofthe binding partners preferably has a suitable marker such as biotin,avidin, lectin, a glycosyl moiety, a specific ligand, or a specificreceptor. The other can be conjugated to the appropriate nanoparticlesas mentioned below. The full sandwich is then captured at the test zonewhere the binding partner of the suitable marker, including, but notlimited to, avidin for biotin, biotin for avidin, glycosyl moiety forlectin, lectin for the glycosyl moiety, a receptor for the ligand, or aligand for the receptor, is immobilized.

FIG. 12A shows an example of a test strip in an embodiment of thepresent invention. The test strip preferably includes an absorbent pad1042, a sample application zone 1044, a detection zone 1052, and anoptional waste pad 1047. The test strip also preferably includes acarrier backing 1048. In this embodiment, the entire sandwich (firstbinding partner 1213-analyte-1040-second binding partner-1218) forms inthe sample application zone 1044. The “full sandwich” 1214 is shown inFIG. 12B. The test zone 1045 in this embodiment includes an immobilizedtag 1210 that binds to the tag 1219 of the second binding partner 1218.The immobilized tag 1210 does not bind directly to the analyte 1040;instead, it binds through an intermediary, the tag 1219 on the secondbinding partner 1218 for the analyte 1040.

In this embodiment, a first binding partner 1213, which is part of thelabeled conjugate 1205, binds the analyte 1040 in the test sample toform half a sandwich. The second binding partner 1218 also includes atag 1219. The second binding partner 1218 in this embodiment ispreferably pre-loaded and dried on the sample application zone 1044 ofthe test strip, while the labeled conjugate 1205 is preferablypre-loaded and dried onto a labeled conjugate zone 1215 upstream of thesample application zone 1044. Alternatively, the second binding partner1218 and/or the labeled conjugate zone 1215 may be located anywhere onthe test strip upstream of the detection zone 1052 including, but notlimited to, overlapping the sample application zone 1044, upstream ofthe sample application zone 1044, or between the sample application zone1044 and the detection zone 1052. In one preferred embodiment,approximately 75-80% of the labeled 1209 conjugate 1205 is upstream ofthe sample application zone (with approximately 20-25% of the labeledconjugate 1205 overlapping the sample application zone 1044) andapproximately 75-80% of the second binding partner 1218 is locateddownstream of the sample application zone 1044 (with approximately20-25% of the second binding partner overlapping the sample applicationzone 44). Although not preferred, in other embodiments, either thelabeled conjugate 1205, the second binding partner 1218, or both may belocated in the buffer or pre-mixed with the sample before the sample isadded to the test strip. In still other embodiments, any or all of thecomponents overlap the detection zone 1052.

In some embodiments, both the first binding partner 1213 and the secondbinding partner 1218 are different antibodies to the analyte 1040. Inother embodiments, specific binding partners may also be antigenscapable of binding to antibodies against the analyte. Other types ofbinding partners are bioorganic macromolecules like aptamers orreceptors, nanoparticles or nucleic acids. The device shown in FIG. 12Acan be used for any binding assays, and can avoid the use ofantibody/antigens or nucleic acids, for example, in ligand-receptorbinding assays and enzyme substrate binding assays.

In one preferred embodiment, the second binding partner 1218 is tagged1219 with biotin. In embodiments where the tag 1219 on the secondbinding partner 1218 is biotin, the immobilized tag 1210 in thedetection zone 1052 is preferably avidin, neutravidin, or streptavidin.In other embodiments, the second binding partner 1218 is tagged 1219with avidin, neutravidin, or streptavidin. In these embodiments, theimmobilized tag 1210 in the detection zone 1052 is preferably biotin.Alternatively, the tag 1219 on the second binding partner 1218 may be alectin and the immobilized tag 1210 may be a glycosyl moiety. Forexample, in some embodiments, the lectin is the Garden pea Lectin andthe glycosyl moiety is an erythrocyte glycosyl unit. The tag on thesecond binding partner and the immobilized tag may be reversed withinthe spirit of the present invention. For example, the glycosyl moietymay be the tag on the second binding partner, with an immobilized lectintag in the detection zone. In other embodiments, other receptors andligands may be used for the tags.

In operation, a sample collector containing the sample is placed suchthat the sample is directly above the sample application zone 1044. Inpreferred embodiments, the sample has not been subject to pretreatmentprior to application to the test strip. Instead, the sample is still inits native form.

The sample is transferred to the sample application zone 1044 of thetest strip. A sandwich forms with the labeled conjugate 1205 as onepiece of bread and the second binding partner 1218 as a second piece ofbread, with the analyte 1040 in between them, when the three componentscome into contact with each other during flow 1043. The labeledconjugate 1205-analyte 1040 (if present)-second binding partner 1218complex (a complete sandwich) flow to the detection zone 1052. Animmobilized tag 1210 in the test zone 1045 then binds the tag 1219.Since the labeled conjugate 1205 includes a label 1209, the complex thatforms is detectable and indicates a positive result. Proper operation ofthe test also results in a detectable positive result in the controlzone 1046, preferably due to the interaction between a control linebinding partner and its immobilized partner in the control zone 1046.

One or more lysis or mucolytic agents may be located (for example, driedon or otherwise included in the membranes making up these zones) in anyof various locations on the test strip, including within the absorbentpad 1042, the labeled conjugate zone 1215, the sample application zone1044, in the detection zone 1052, or overlapping any of theintersections between these areas on the strip. One or more lysis ormucolytic agents may alternatively be located in the buffer. In otherembodiments, the lysis or mucolytic agents may be on the samplecollector as long as the lysis or mucolytic agent being used isbiocompatible. In yet another embodiment, one or more lysis or mucolyticagents may be placed where the sample application zone 1044 meets thedetection zone 1052. In preferred embodiments, the lysis or mucolyticagent overlaps or is upstream of the sample application zone 1044. Ifthere are multiple lysis and/or mucolytic agents, they can be located inthe same or different locations on the test strip or on the samplecollector. A single lysis and/or mucolytic agent could be located inmore than one location, if desired.

In some preferred embodiments using tags, the detection zone includes anantibody against the tag. The antibody may be a monoclonal, polyclonalor single domain antibody. For example, when the tag is biotin, ananti-biotin antibody is immobilized in the test zone instead of avidin,neutravidin, or streptavidin.

FIGS. 13A through 13C show an example of an embodiment of the systemwith a sample compressor 1030, a sample collector 1035, and a sampleanalysis device (a test strip in the figure). Similar to FIG. 11A-11C,the test strip preferably includes an absorbent pad 1042, a sampleapplication zone 1044, a detection zone 1052, and an optional waste pad1047. The test strip also preferably includes a carrier backing 1048. Inthis embodiment, the entire sandwich (first binding partner1037-analyte-1040-second binding partner-1038) forms in the sampleapplication zone 1044 (preferably before the addition of buffer). Insome embodiments, placement of the sample collector 1035 above thesample application zone 1044 is not simultaneous with placement of thesample compressor 1030. In other words, in these embodiments, some ofthe sample is transferred to the sample application zone 1044 before thesample compressor 1030 is added to the vertical stack.

The test zone 1045 in this embodiment includes an immobilized tag 1050that binds to the tag 1039 of the second binding partner 1038. In thisembodiment, a first binding partner 1037, which is part of the conjugate1036 and is preferably pre-loaded and dried on the pad 1033 of thesample compressor 1030, binds the analyte 1040 in the test sample toform a half sandwich. The second binding partner 1038 in this embodimentis also preferably pre-loaded and dried on the pad 1033 of the samplecompressor. The second binding partner 1038 also includes a tag 1039.

The full sandwich 1320 that forms between the binding partner 1037 ofthe conjugate 1036, the analyte 1040, and the second binding partner1038 in this embodiment (as well as the embodiments in FIGS. 14A-14B,15A-15B, 16B, 16C, and 16D) is shown in FIG. 13B. In preferredembodiments, the pad 1033 on the sample compressor 1030 also includes acontrol zone binding partner 1061 (shown in FIG. 11C) with a detectablelabel. The control zone binding partner 1061 complexes with its bindingpartner in the control zone 1046. Including the control zone bindingpartner 1061 on the sample compressor 1030, instead of on the test stripor in the buffer as known in the prior art, permits the user to be surethat the components on the sample compressor 1030, which include boththe conjugate 1036 and the control zone binding partner 1061, haveeffectively transferred to the sample analysis device and thus ensuresproper operation of the system.

In one example, both the first binding partner 1037 and the secondbinding partner 1038 are different antibodies to the analyte. Thecontrol zone binding partner 1061 is also preferably an antibody, andits binding partner at the control zone is an antigen (or vice versa).In other embodiments, specific binding partners may also be antigenscapable of binding to antibodies against the analyte. Other types ofbinding partners are bioorganic macromolecules like aptamers orreceptors, nanoparticles, or nucleic acids. The device shown in FIGS.13A-13C of the present invention can be used for any binding assays, andcan avoid the use of antibody/antigens or nucleic acids, for example, inligand-receptor binding assays and enzyme-substrate binding assays.

In one preferred embodiment, the second binding partner 1038 is taggedwith biotin 1039. In embodiments where the tag 1039 on the secondbinding partner 1038 is biotin, the immobilized tag 1050 in thedetection zone is preferably avidin, neutravidin, or streptavidin. Inother embodiments, the second binding partner 1038 is tagged 1039 withavidin, neutravidin, or streptavidin. In these embodiments, theimmobilized tag 1150 in the detection zone 1052 is preferably biotin.Alternatively, the tag 1039 on the second binding partner 1038 may be alectin and the immobilized tag 1050 may be a glycosyl moiety. Forexample, in some embodiments, the lectin is the Garden pea Lectin andthe glycosyl moiety is an erythrocyte glycosyl unit. The tag on thesecond binding partner and the immobilized tag may be reversed withinthe spirit of the present invention. For example, the glycosyl moietymay be the tag on the second binding partner, with an immobilized lectintag in the detection zone. In other embodiments, other receptors andligands may be used for the tags.

In operation, the sample collector 1035 is placed such that the sampleis directly above the sample application zone 1044. The samplecompressor 1030 exerts pressure 1051 on the sample collector 1035. Thepressure transfers the sample (including the analyte 1040, if present),the conjugate 1036, and the tagged second binding partner 1038 onto thesample application zone 1044. If there is also a control zone bindingpartner 1061 on the sample compressor 1030, the control zone bindingpartner 1061 is also transferred. Note that the transfer is due topressure, not due to flow or capillary action. Then, buffer 1043 isadded to permit flow of the conjugate 1036-analyte 1040 (ifpresent)-second binding partner 1038 complex (a complete sandwich) tothe detection zone 1052. An immobilized tag 1050 in the test zone 1045then binds the tag 1039. Since the conjugate 1036 includes a label 1041,the complex that forms is detectable and indicates a positive result.Proper operation of the test also results in a detectable positiveresult in the control zone 1046 due to the interaction between thecontrol zone binding partner 1061 and its immobilized partner in thecontrol zone 1046.

One or more lysis or mucolytic agents may be located (for example, driedon or otherwise included in the membranes making up these zones) on thetest strip in various locations including, in the absorbent pad 1042, inthe sample application zone 1044, directly upstream of the detectionzone 1052, in the detection zone itself, or within any of theintersections between these sections of the test strip. Alternatively,one or more lysis or mucolytic agents is pre-loaded and dried on thesample compressor 1030. One or more lysis or mucolytic agents mayalternatively be located in the buffer. In other embodiments, the lysisor mucolytic agents may be on the sample collector as long as the lysisor mucolytic agent being used is biocompatible. In yet anotherembodiment, one or more lysis or mucolytic agents may be placed wherethe sample application zone 1044 meets the detection zone 1052 or wherethe sample application zone meets the nitrocellulose membrane. Inpreferred embodiments, the lysis or mucolytic agent overlaps or isupstream of the sample application zone 1044 or is located on the samplecompressor 1030. If there are multiple lysis and/or mucolytic agents,they can be located in the same or different locations on the teststrip, on the sample collector, or on the sample compressor. A singlelysis and/or mucolytic agent could be located in more than one location,if desired.

In another embodiment, the two binding partners for the analyte arelocated in such a way to achieve a “vertical sandwich” where the samplebinds with the conjugate being compressed from the second plane and canbind simultaneously or concurrently with the other binding partnerlocated on the strip in the plane of the strip. Thus a sandwiching ofthe analyte in the sample is achieved by binding to the partner from theconjugate delivered from above the plane of the strip and binding to thesecond binding partner located on the plane of the strip below thesample delivering material.

FIGS. 14A and 14B show another example of an embodiment of the systemwith a sample compressor 1030, a sample collector 1035, and a sampleanalysis device (a test strip in the figure). Similar to FIG. 11A-11C,the test strip preferably includes an absorbent pad 1042, a sampleapplication zone 1044, a detection zone 1052, and an optional waste pad1047. The test strip also preferably includes a carrier backing 1048.Similar to the embodiment shown in FIGS. 13A and 13C, in thisembodiment, the entire sandwich (first binding partner 1037-analyte1040-second binding partner 1038) forms in the sample application zone1044. The test zone 1045 in this embodiment includes an immobilized tag1050 that binds to the tag 1039 of the second binding partner 1038. Inthis embodiment, a first binding partner 1037, which is part of theconjugate 1036 and is preferably pre-loaded and dried on the pad 1033 ofthe sample compressor 1030, binds the analyte 1040 in the test sample toform a half sandwich. The second binding partner 1038 in this embodimentis preferably pre-loaded and dried on the sample application zone 1044of the test strip. The second binding partner 1038 also includes a tag1039. Alternatively, the second binding partner 1038 in this embodimentmay be located anywhere on the test strip upstream of the detection zoneincluding, but not limited to, overlapping the sample application zone,upstream of the sample application zone, and between the sampleapplication zone and the detection zone.

In preferred embodiments, the pad 1033 on the sample compressor 1030also includes a control zone binding partner 1061 (shown in FIG. 11C)with a detectable label. The control zone binding partner 1061 complexeswith its binding partner in the control zone 1046. Including the controlzone binding partner 1061 on the sample compressor 1030, instead of onthe test strip or in the buffer as known in the prior art, permits theuser to be sure that the components on the sample compressor 1030, whichinclude both the conjugate 1036 and the control zone binding partner1061, have effectively transferred to the sample analysis device andthus ensures proper operation of the system.

In one example, both the first binding partner 1037 and the secondbinding partner 1038 are different antibodies to the analyte. Thecontrol zone binding partner 1061 is also preferably an antibody, andits binding partner at the control zone is an antigen (or vice versa).In other embodiments, specific binding partners may also be antigenscapable of binding to antibodies against the analyte. Other types ofbinding partners are bioorganic macromolecules like aptamers orreceptors, nanoparticles, or nucleic acids. The device shown in FIGS.14A-14B of the present invention can be used for any binding assays, andcan avoid the use of antibody/antigens or nucleic acids, for example, inligand-receptor binding assays and enzyme-substrate binding assays.

In one preferred embodiment, the second binding partner 1038 is taggedwith biotin 1039. In embodiments where the tag 1039 on the secondbinding partner 1038 is biotin, the immobilized tag 1050 in thedetection zone is preferably avidin, neutravidin, or streptavidin. Inother embodiments, the second binding partner 1038 is tagged 1039 withavidin, neutravidin, or streptavidin. In these embodiments, theimmobilized tag 1050 in the detection zone 1052 is preferably biotin.Alternatively, the tag 1039 on the second binding partner 1038 may be alectin and the immobilized tag 1050 may be a glycosyl moiety. Forexample, in some embodiments, the lectin is the Garden pea Lectin andthe glycosyl moiety is an erythrocyte glycosyl unit. The tag on thesecond binding partner and the immobilized tag may be reversed withinthe spirit of the present invention. For example, the glycosyl moietymay be the tag on the second binding partner, with an immobilized lectintag in the detection zone. In other embodiments, other receptors andligands may be used for the tags.

In operation, the sample collector 1035 is placed such that the sampleis directly above the sample application zone 1044. The samplecompressor 1030 exerts pressure 1051 on the sample collector 1035, usingpressure to transfer the sample (including the analyte 1040, if present)and the conjugate 1036 onto the sample application zone 1044. A“vertical” sandwich forms with the conjugate 1036 as the top piece andthe second binding partner 1038 as the bottom piece, with the analyte1040 in between them. If there is also a control zone binding partner1061 on the sample compressor 1030, the control zone binding partner1061 is also transferred. Note that the transfer is due to pressure, notdue to flow or capillary action. Then, buffer 1043 is added to permitflow of the conjugate 1036-analyte 1040 (if present)-second bindingpartner 1038 complex (a complete sandwich) to the detection zone 1052.An immobilized tag 1050 in the test zone 1045 then binds the tag 1039.Since the conjugate 1036 includes a label 1041, the complex that formsis detectable and indicates a positive result. Proper operation of thetest also results in a detectable positive result in the control zone1046 due to the interaction between the control zone binding partner1061 and its immobilized partner in the control zone 1046.

One or more lysis or mucolytic agents may be located (for example, driedon or otherwise included in the membranes making up these zones) on thetest strip in various locations including, in the absorbent pad 1042, inthe sample application zone 1044, directly upstream of the detectionzone 1052, in the detection zone itself, or within any of theintersections between these sections of the test strip. Alternatively,one or more lysis or mucolytic agents is pre-loaded and dried on thesample compressor 1030. One or more lysis or mucolytic agents mayalternatively be located in the buffer. In other embodiments, the lysisor mucolytic agents may be on the sample collector as long as the lysisor mucolytic agent being used is biocompatible. In yet anotherembodiment, one or more lysis or mucolytic agents may be placed wherethe sample application zone 1044 meets the detection zone 1052 or wherethe sample application zone meets the nitrocellulose membrane. Inpreferred embodiments, the lysis or mucolytic agent overlaps or isupstream of the sample application zone 1044 or is located on the samplecompressor 1030. If there are multiple lysis and/or mucolytic agents,they can be located in the same or different locations on the teststrip, on the sample collector, or on the sample compressor. A singlelysis and/or mucolytic agent could be located in more than one location,if desired.

FIGS. 15A and 15B show another embodiment of the present invention,where the sample compressor 1030 includes the second binding partner1038 for the analyte 1040, coupled with a tag 1039, and the test stripincludes the conjugate 1036, which includes both a first binding partner1037 for the analyte 1040 and a detectable label 1041, and theimmobilized tag 1050 that binds to the tag on the second binding partnerin the test zone 1045. This embodiment operates similarly to theembodiment described with respect to FIGS. 14A and 14B, except that the“vertical” sandwich forms with the second binding partner 1038 as thetop piece and the conjugate 1036 as the bottom piece, with the analyte1040 in between them. Alternatively, the conjugate 1036 in thisembodiment may be located anywhere on the test strip upstream of thedetection zone including, but not limited to, overlapping the sampleapplication zone, upstream of the sample application zone, or betweenthe sample application zone and the detection zone.

One or more lysis or mucolytic agents may be located (for example, driedon or otherwise included in the membranes making up these zones) on thetest strip in various locations including, in the absorbent pad 1042, inthe sample application zone 1044, directly upstream of the detectionzone 1052, in the detection zone itself, or within any of theintersections between these sections of the test strip. Alternatively,one or more lysis or mucolytic agents is pre-loaded and dried on thesample compressor 1030. One or more lysis or mucolytic agents mayalternatively be located in the buffer. In other embodiments, the lysisor mucolytic agents may be on the sample collector as long as the lysisor mucolytic agent being used is biocompatible. In yet anotherembodiment, one or more lysis or mucolytic agents may be placed wherethe sample application zone 1044 meets the detection zone 1052 or wherethe sample application zone meets the nitrocellulose membrane. Inpreferred embodiments, the lysis or mucolytic agent overlaps or isupstream of the sample application zone 1044 or is located on the samplecompressor 1030. If there are multiple lysis and/or mucolytic agents,they can be located in the same or different locations on the teststrip, on the sample collector, or on the sample compressor. A singlelysis and/or mucolytic agent could be located in more than one location,if desired.

FIGS. 16A through 16D are similar to FIGS. 11C, 13C, 14B, and 15B,respectively, except that the detection zone 1052 overlaps the sampleapplication zone 1044 in these figures. The detection zone in theseembodiments is preferably made of nitrocellulose. Although no lateralflow is strictly required to run the assay in these embodiments, atleast a nominal amount of flow is preferred such that the sandwich isable to bind in the test zone and any unbound conjugate is washed out ofthe test zone. In one embodiment, instead of a running buffer beingapplied to an end of the test strip, a washing fluid may be applieddirectly to the test zone, either from above or from the side, forexample using a water bottle. In one embodiment, the sample compressorand the sample collector are substantially transparent so that the testzone can be read without removal of the vertical stack from the teststrip. Note that, while both the test zone 1045 and the control 1046 areshown within the sample application zone in these figures, in otherembodiments the test zone 1045 could overlap the sample application zone1044 while the control zone 1046 is downstream of the sample applicationzone 1044. If the control zone was laterally downstream from the sampleapplication zone 1044, it would be necessary to add buffer to allowflow. In addition, it may be preferable to add a buffer, for example abuffer that includes silver, to enhance the signal from a positiveresult.

One or more lysis or mucolytic agents may be located (for example, driedon or otherwise included in the membranes making up these zones) on thetest strip in various locations including, in the absorbent pad 1042, inthe sample application zone 1044, overlapping or in the detection zone,or within any of the intersections between these sections of the teststrip. Alternatively, one or more lysis or mucolytic agents ispre-loaded and dried on the sample compressor 1030. One or more lysis ormucolytic agents may alternatively be located in the buffer. In otherembodiments, the lysis or mucolytic agents may be on the samplecollector as long as the lysis or mucolytic agent being used isbiocompatible. In yet another embodiment, one or more lysis or mucolyticagents may be placed where the sample application zone 1044 meets thedetection zone 1052 or where the sample application zone meets thenitrocellulose membrane. In preferred embodiments, the lysis ormucolytic agent overlaps or is upstream of the sample application zone1044 or is located on the sample compressor 1030. If there are multiplelysis and/or mucolytic agents, they can be located in the same ordifferent locations on the test strip, on the sample collector, or onthe sample compressor. A single lysis and/or mucolytic agent could belocated in more than one location, if desired.

A universal test strip 1780, as shown in FIG. 17A, may be used when thesample compressor 1030 includes both of the binding partners 1037, 1038for the analyte 1040. The sample compressor 1030 and the samplecollector 1035 would be transferred to the universal test strip 1780 atthe sample window 1781. Since the elements specific to the analyte 1040being tested are on the sample compressor 1030, the test zone 1783 inthe viewing window 1782 of the universal test strip 1780 only needs tohave a tag 1050 that complexes with the tag 1039 on the second bindingpartner 1038 for the analyte 1040. For example, when the second bindingpartner 1038 for the analyte 1040 is tagged 1039 with biotin, the testzone 1783 of the universal test strip 1780 would include avidin 1039, abinding partner for biotin. The universal test strip 1780 alsopreferably includes a control zone 1784 and a housing 1785. For theembodiments of FIGS. 16A through 16D, the test zone is located in thesample window 1781. In other embodiments, the suitable marker can be anucleotide sequence that can hybridize with the suitable nucleic acidsequence immobilized at the test zone.

One or more lysis or mucolytic agents may be located (for example, driedon or otherwise included in the membranes making up these zones)anywhere on the universal test strip 1780 where it would come intocontact with the sample and effectively break down the mucous membranesor otherwise lyse the sample. In one preferred embodiment with auniversal test strip 1780, one or more lysis or mucolytic agents ispre-loaded and dried onto the pad of the sample compressor 1030. One ormore lysis or mucolytic agents may alternatively be located in thebuffer. In other embodiments, the lysis or mucolytic agents may be onthe sample collector 1060 as long as the lysis or mucolytic agent beingused is biocompatible. In preferred embodiments where the lysis ormucolytic agents are located on the universal test strip 1780, the lysisor mucolytic agent overlaps or is upstream of the location where thesample collector 1035 contacts the test strip 1780 (for example upstreamof the sample window 1781). If there are multiple lysis and/or mucolyticagents, they can be located in the same or different locations on thetest strip, on the sample collector, or on the sample compressor. Asingle lysis and/or mucolytic agent could be located in more than onelocation, if desired.

Although the sample compressor and the sample collector are shown asseparate entities in FIGS. 9-17A, the pad 1033 of the sample compressorand the sample collector portion 1060 of the sample collector may becomponents of a single element within the spirit of the presentinvention. For example, the sample collector may be rotatably orflexibly or connected as part of a cartridge to the sample compressor,such that a sample can be collected from a patient with the samplecollection portion without exposing the patient to the sample compressorpad and then the sample collection portion and sample compressor pad canbe brought into contact for application to the sample application zoneof the test strip by compression. The sample collector also may berotatably or flexibly connected to the test cassette or may be insertedas a cartridge. In another embodiment, the sample may be forciblyinjected directly onto the test strip prior to placing the compressorand/or conjugates into position. In yet another embodiment, the samplecollector may contact the conjugates in an external cartridge that thensnaps or inserts into a test cassette to bring the material in contactwith the test strip.

In some embodiments, the sample compressor 1030 is rotatably connectedto the housing 1785 as shown in FIG. 17B. While the hinge of the samplecompressor 1030 is shown such that the sample compressor 1030 is rotatedtowards the downstream end of the strip when open, the housing could bedesigned such that the sample compressor 1030 is hinged to either sideor in other directions within the spirit of the present invention. Thesample collection portion 1060 of the sample collector 1035 ispreferably inserted from the side such that it lines up with aninsertion hole 1788 on the side of the housing 1785. However, the samplecollector 1035 could be inserted in any direction depending upon thedesign of the housing. The sample compressor 1030 preferably includes apad (not visible in FIG. 17B), with one or more assay components,located on the surface of the sample compressor facing the sampleapplication zone of the test strip 1780. The sample compressor 1030 isthen closed such that a compression pressure is applied to the verticalstack of the pad of the sample compressor, the sample collectionportion, and the sample application zone to transfer the sample and theone or more assay components to the sample application zone of the teststrip. While there is an absorbent pad sticking out of the housing atthe far upstream end of the device in FIG. 17B, the length of theabsorbent pad may vary. In fact, as long as buffer can be added at theupstream end (for example, through an application window in thehousing), it is not necessary to have the absorbent pad extendsignificantly outside the housing. In this embodiment, there is nopossibility of losing the sample compressor, and there is no need toalign the sample compressor with the sample application zone whenforming the vertical stack. One advantage of these embodiments is thatthey allow for a time lapse between sample application and the actualinitiation of flow to the test zone. In other words, the sandwich can bepre-made, and the flow initiated much later.

Alternatively, the pad 1033 may be separate from the sample compressorwithin the spirit of the present invention. The pad may be on a bindingpartner applicator similar to the sample collector. In theseembodiments, the binding partner applicator may be located between thesample collection portion and the sample application zone when thepressure is applied by the sample compressor to transfer the sample tothe sample application zone.

FIG. 18 shows a vertical stack including a sample compressor 1030, asample collector 1035 with a sample collection portion 1060, a bindingpartner applicator 1862 with an applicator pad 1864, and a sampleapplication zone 1044 of a test strip. While the binding partnerapplicator 1862 includes a handle in FIG. 18, the binding partnerapplicator 1862 could alternatively simply be a pad. The ledge portion1034 of the sample compressor 1030 applies pressure to the samplecollection portion 1060 loaded with a sample and the applicator pad 1864loaded with at least one binding partner for an analyte to be tested forin the sample. The pressure preferably forces at least a portion of thesample from the sample collection portion 1060 to wet the applicator pad1864, thereby mobilizing some of the binding partner such that at leastsome of the sample and some of the binding partner are transferred tothe sample application zone 1044. In some embodiments, this transferoccurs without dilution. In embodiments with small sample volumes orviscous or solid samples, however, an additional liquid may be used tofacilitate transfer of the sample and the binding partner to the teststrip. In some embodiments, as shown in FIG. 18, the sample compressorhas no pad, although a pad may be used to aid in transfer, such as bysupplying additional liquid or buffer, within the spirit of the presentinvention. In some embodiments, as shown in FIG. 18, the samplecollection portion 1060 is located between the sample compressor 1030and the applicator pad 1864 in the vertical stack to aid in transfer ofthe binding partner to the test strip during compression. Alternatively,the applicator pad 1864 may be placed between the sample compressor 1030and the sample collection portion 1060 within the spirit of the presentinvention. In embodiments where the full sandwich forms prior toreaching the test zone, two binding partner applicators (a separateapplicator for each binding partner of the analyte) may be used, withthe sample collection portion, the first applicator pad, and the secondapplicator pad being placed in any order on the vertical stack withinthe spirit of the present invention. Alternatively, a single bindingpartner applicator could include both of the binding partners for theanalyte. In other embodiments, the sample, the first binding partner,and the second binding partner may be applied sequentially to the teststrip in any order using the sample compressor within the spirit of thepresent invention.

In a method of applying a sample to a test strip of a lateral flowdevice, at least one external binding partner is first placed on thesample application zone of the test strip. The external binding partnermay be located on an external pad. In embodiments where there are twoanalyte binding partners that bind the analyte prior to reaching thetest zone, either one or both of the analyte binding partners may beadded. A sample collector that includes the sample is placed in avertical stack between the external binding partner and a samplecompressor. The sample compressor applies pressure to the samplecollector to transfer the external binding partner and at least aportion of the sample to the sample application zone. Alternatively, theexternal binding partner could be added and compressed by the samplecompressor, then removed, before the sample collector is stacked abovethe sample application zone, where the sample is compressed onto thetest strip. In another alternative embodiment, at least one externalbinding partner is placed in the vertical stack between the samplecompressor and sample collector. Alternatively, the sample collector isadded and compressed, then removed, and then the external bindingpartner is added and compressed onto the test strip. In otherembodiments, the sample collector is in a vertical stack between a firstexternal binding partner and a second external binding partner, and thesample compressor applies pressure to the vertical stack. In theseembodiments, neither the strip nor the sample compressor has a specificanalyte binding partner. The sample, the analyte binding partner, andthe mobile control binding partner may also be applied to the sampleapplication zone in multiple steps in any combination within the spiritof the present invention.

Alternatively, in a lateral flow device of the present invention, thesample compressor may be a universal sample compressor with nocomponents specific to the analyte of interest. In one embodiment, thesample compressor contains no components of the assay. In embodimentswith a control, the pad of the sample compressor contains only themobile control zone binding partner. In these embodiments, one or morebinding partner applicators include at least one binding partner for theanalyte and become part of the vertical stack with the sample compressorand the sample collector when the sample is transferred to the sampleapplication zone. The sample, the analyte binding partner, and themobile control binding partner may also be applied to the sampleapplication zone in multiple steps in any combination within the spiritof the present invention. In some embodiments, one or more lysis ormucolytic agents is also pre-loaded and dried onto the universal samplecompressor.

In another embodiment of the present invention, the sample compressor1030 also serves as the sample collector, and the pad 1033 of the samplecompressor also serves as the sample collection portion. In thisembodiment, the conjugate, the second binding partner, the control linebinding partner, and/or any combination of the three, are preferablylocated on a back surface of the pad 1033, where the pad is attached tothe sample compressor arm. In embodiments where sample collection needsto be performed sterilely, the sample compressor 1030 is then preferablysterilized by radiation prior to use as a sample collector. The sampleis then collected using the front part of the pad so that the patient isnot exposed to the conjugate or the second binding partner during sampleacquisition. When the sample is applied to the sample application zoneof the test strip, the pad is preferably compressed so that the samplemixes with the conjugate or the second binding partner and at least aportion of both is squeezed out onto the test strip. There mayoptionally be one or more lysis or mucolytic agents on the combinationsample compressor/sample collector. If the lysis or mucolytic agent isbiocompatible, the lysis or mucolytic agent can be located either on thefront part or the back surface of the pad 1033. In other embodiments,the lysis or mucolytic agent is located on the back surface of the pad1033.

The analytical tests discussed herein preferably permit a result whilethe patient is still being examined by the practitioner. The results ofthe tests are preferably determined within 20 minutes of transferringthe sample to the device. In a preferred embodiment, the test result isobtained in 10 minutes or less after applying the sample to the device,and it is preferably read at approximately 10 minutes. In samples thatare highly positive, a readout of the test zone (preferably a test line)is visible within approximately 1-5 minutes.

In some embodiments, the devices and methods of the present inventiondetect nucleic acids in a sample without the use of an amplificationstep for the target nucleic acid. In some embodiments, the detectednucleic acids are also quantified. The lateral flow detector may be usedto detect a target nucleic acid sequence associated with any targetvirus, bacterium, fungus, or other pathogen, any genetic deficiency, orany other target nucleic acid in a sample. The target nucleic acid maybe any nucleic acid including, but not limited to, DNA, anoligonucleotide, messenger RNA, or any other type of RNA. The assay ispreferably run within a matter of minutes to a few hours after thesample is obtained, but the assay may be run at a later time such as atleast 24 hours after obtaining the sample. The flow of the transportliquid in the detector may be gravity-dependent or as a result ofcapillary action or surface tension. The transport liquid may be appliedby dipping the test strip in the transport liquid or the transportliquid may be contained in a test housing for the test strip.

A lateral flow nucleic acid detector in these embodiments may beuniplanar with a single sheet on a test strip for the detection zone.Alternatively, the detector may be multiplanar with multiple detectionzones on multiple sheets in fluid communication for simultaneous assaysfor the same or different target nucleic acids from the same ordifferent samples.

A sample for testing in these embodiments may be any sample expected topotentially include a target nucleic acid including, but not limited to,saliva, tears, cerebral spinal fluid, skin lesions, vaginal fluid,penile fluid, mucus, tissue, blood, urine, an environmental watersample, and a soil sample. In most cases, it is preferable to add adenaturant, lysis or mucolytic agent in situ to the sample in order tomake the nucleic acids in the sample accessible to the first and secondcomplexes. The denaturant, lysis or mucolytic agent is preferablypre-loaded onto a zone of the test strip or onto a sample compressor sothat the sample may be applied directly to the test strip without a stepof adding denaturant, lysis or mucolytic agent. The denaturant, lysis ormucolytic agent is pre-loaded onto the test strip or on the samplecompressor in a location so that it frees the nucleic acids prior to thesample reaching the first complex on the test strip. The denaturant,lysis or mucolytic agent is preferably soluble or miscible in thetransport liquid and located in the sample application zone or betweenthe sample application zone and the zone where the first complex ispre-loaded.

In some embodiments, the sensitivity of visually read lateral flow assaytests is enhanced by adding a small quantity of fluorescing dye orfluorescing latex bead conjugates to the initial conjugate material.When the visible spectrum test line is visibly present, the test resultis observed and recorded. However, in the case of weak positives that donot give rise to a distinct visual test line, a light of an appropriatespectrum, such as a UV spectrum, is cast on the test line to excite andfluoresce the fluorescing latex beads which are bound in the test lineto enhance the visible color at the test line.

In some embodiments, the present invention provides a lateral flow assaythat uses the lysis or mucolytic zone to help differentiate viral andbacterial infections. One situation where a lysis agent improves assayefficiency is in assaying for the presence of Human MxA, a 78 kDaprotein which accumulates in the cytoplasm as a response to viralinfection. The presence of this protein can help to distinguish betweenbacterial and viral infection in febrile children. In situ lysis using acombination of 1% to 6% weight/volume CHAPS and 0.5% to 2% weight/volumeNP40 as the lysis agent improves detection of MxA in fresh or frozenwhole blood.

A combined point of care diagnostic device tests markers for both viraland bacterial infection, and can effectively assist in the rapiddifferentiation of viral and bacterial infections, for example at theoutpatient office or during an urgent care visit. This ability candramatically reduce health care costs by limiting misdiagnosis and thesubsequent overuse of antibiotics. Such a practice may limit antibioticallergies, adverse events, and antibiotic resistance. The rapid resultobtained from the test also permits a result while the patient is stillbeing examined by the practitioner.

In one preferred embodiment, the marker for viral infection is MxA andthe marker for bacterial infection is C-reactive protein (CRP). High MxAprotein levels are strongly correlated with systemic viral infection andincreased CRP is more associated with bacterial infections. The presentinvention includes a rapid infectious screening test for identifying MxAand CRP in samples. MxA is present in leukocytes (white blood cells).Therefore, the sample can be taken anywhere leukocytes are available,for example in a peripheral blood sample, nasopharyngeal aspirates,tears, spinal fluid, and middle ear aspirates.

In other embodiments, other markers for viral infection and/or bacterialinfection may be used. For example, approximately 12% of host genesalter their expression after Lymphocytic Choriomeningitis Virus (LCMV)infection, and a subset of these genes can discriminate between virulentand nonvirulent LCMV infection. Major transcription changes have beengiven preliminary confirmation by quantitative PCR and protein studiesand are potentially valuable candidates as biomarkers for arenavirusdisease. Other markers for bacterial infection include, but are notlimited to, procalcitonin, urinary trypsin inhibitor (uTi),lipopolysaccharide, IL-1, IL-6, IL-8, IL-10, ESR and an elevated WBCcount (increased bands), Lactate, Troponin, vascular endothelial growthfactor, platelet derived growth factor, cortisol, proadrenomedullin,macrophage migratory inhibitory marker, activated protein C, CD 4, 8,13, 14, or 64, caspase, placenta derived growth factor, calcitoningene-related peptide, high mobility group 1, copeptin, naturieticpeptides, lipopolysaccharide binding protein, tumor necrosis factoralpha, circulating endothelial progenitor cells, complement 3a, andtriggering receptor expressed on myeloid cells (trem-1).

In one embodiment, the infections being distinguished are respiratoryinfections. In other embodiments, other types of infections, which canbe bacterial or viral, are differentiated using the system of thepresent invention. Some examples include, but are not limited to,encephalitis, meningitis, gastroenteritis, febrile respiratory illness(including bronchitis, pharyngitis, pneumonia), sinusitis, otitis media,urinary tract infections, and conjunctivitis.

The mucolytic agent may be in the running buffer that initiates theassay. In these embodiments, the mucolytic agent lyses the membranousmaterial as it traverses through the strip.

In any of the embodiments described herein, the conjugate, for examplegold or color dyed latex beads, the analyte binding partners, or thetags, can be treated with a mucolytic agent, for example NAC. Bothmobile as well as immobile reagents or zones can be treated withmucolytic agents separately or in combination. These embodiments providemore opportunity for mucolysis.

The mucolytic agent may be localized in any position on the lateral flowtest strip relative to the sample application zone, in the buffer, or onthe sample collector or sample compressor device. In some embodiments,the mucolytic agent may be placed so that it is directly below thesample collector when the sample collector is applied to the lateralflow test strip. In some embodiments, the mucolytic agent may belocalized at the end of the sample pad, or at the junction of the sampleand/or conjugate pad and the detection zone membrane (such as anitrocellulose membrane). The mucolytic agent may alternatively bedeposited at the base of the detection zone membrane (such as anitrocellulose membrane) where the sample and the conjugate complex canencounter it, resulting in further lysis of the sample in the complex.In embodiments with a sample compressor, the mucolytic agent may belocated on the lateral flow test strip, the sample compressor, thesample collector, or in the buffer. The mucolytic agent in any of theseembodiments could be added after the start of the assay.

In another embodiment, the sample collector collects the sample, andthen is placed on the lateral flow test strip (or in the cassette inembodiments where a cassette is used). At that time, the mucolytic agentmay be added to the sample collector and the strip. For example, one ortwo drops of the mucolytic agent may be added at this time. Inembodiments with a sample compressor, this step occurs before the samplecompressor compresses the sample collector. In one example where thetarget is MxA, this method would permit a blood sample to be lysed torelease the leukocytic MxA. This method could be used in this example inany of the embodiments disclosed herein, with or without the use of asample compressor.

In one preferred embodiment, the conjugate zone is upstream of thesample application zone and the sample being tested is blood. In thisembodiment, the mucolytic agent could mucolyse the blood cells(e.g.—erythrocytes, leukocytes, platelets) and also make “holes” in theblood through which the microspheres of the conjugate can go throughunhindered. Intact cells may hinder the movement of nanoparticles ormicrospheres of the conjugate going through a zone where they arelocated. When the mucolytic agent makes “holes” in those intact cells,the smaller nanoparticles can “go through” those holes.

EXAMPLE

One or more lysis agents are dried onto the sample application zone of alateral flow strip. On a per strip basis, the lysis agent is made ofapproximately 2 microliters of 100 mM HEPES buffer (pH 8.0) containing5% CHAP S and 2% NP-40 with 150 mM Sodium Chloride, 0.1% BSA, and 0.1%Sodium Azide (all percentages weight/volume). Up to 10 microliters ofwhole blood are then added to the sample application zone to be lysed insitu. MxA protein is released from inside white blood cells to reactwith an MxA monoclonal antibody on a visual tag (colloidal gold orvisible latex beads). This complex traverses with a running buffercontaining Triton X-100 and is captured by MxA monoclonal antibodiesimmobilized at the test line of the nitrocellulose membrane. Thisbinding at the test line gives rise to a visible indication.

Accordingly, it is to be understood that the embodiments of theinvention herein described are merely illustrative of the application ofthe principles of the invention. Reference herein to details of theillustrated embodiments is not intended to limit the scope of theclaims, which themselves recite those features regarded as essential tothe invention.

1-25. (canceled)
 26. A device for the detection of a target in a samplecomprising; a) a sample application zone for applying a sample to thedevice; b) a detection zone located downstream of the sample applicationzone; and c) a conjugate zone comprising at least one labeled bindingpartner that is able to migrate with an elution medium, wherein theconjugate zone is located on the device at a location such that thesample encounters the labeled binding partner during an assay run on thedevice; and wherein the elution medium comprises at least one mucolyticagent.
 27. The device of claim 26, wherein the conjugate zone is locatedbetween the sample application zone and the detection zone. 28-30.(canceled)
 31. A lateral flow device for detecting an analyte in asample comprising: a sample compressor comprising at least one mucolyticagent; a sample collector comprising a sample collection portion forcollection of the sample; a test strip comprising a sample applicationzone and a test zone; a conjugate comprising a first binding partner forthe analyte and a label; and a second binding partner for the analyte;wherein a component selected from the group consisting of the conjugate,the second binding partner and both the conjugate and the second bindingpartner is not located on the test strip prior to use of the lateralflow device; and wherein the sample compressor, the sample collector,and the test strip form a vertical stack to apply the sample to the teststrip by compression; wherein the sample collector is located betweenthe sample compressor and the test strip in the vertical stack.
 32. Amethod of applying a sample to a test strip of a lateral flow device,the method comprising the steps of: a) placing a sample collectorcomprising a sample collection portion with the sample in a verticalstack between a sample compressor and a sample application zone of thetest strip, wherein the sample compressor comprises at least onemucolytic agent; and b) applying a pressure to the sample collectionportion using the sample compressor to transfer at least a portion ofthe sample to the sample application zone.
 33. A lateral flow device fordetecting an analyte in a sample comprising: a sample compressor; asample collector comprising a sample collection portion for collectionof the sample; a test strip comprising a sample application zone and atest zone; a conjugate comprising a first binding partner for theanalyte and a label; a second binding partner for the analyte; and amucolytic agent located in a location selected from the group consistingof: i) on the sample compressor; ii) on the sample collection portion ofthe sample collector and iii) on the test strip; wherein a componentselected from the group consisting of the conjugate, the second bindingpartner and both the conjugate and the second binding partner is notlocated on the test strip prior to use of the lateral flow device; andwherein the sample compressor, the sample collector, and the componentnot on the test strip prior to use of the lateral flow device form avertical stack to apply the sample to the test strip by compression. 34.The lateral flow device of claim 33, further comprising at least onelysis agent.
 35. (canceled)